This invention relates to the expression of non-infectious, non-replicating immunogenic HIV-like particles and, in particular, to genetic modifications required to obtain long term high level constitutive expression of such particles.
Human immunodeficiency virus is a human retrovirus and is the etiological agent of acquired immunodeficiency syndrome (AIDS). It is estimated that more than 18 million people have been infected with HIV as of mid 1996 (ref. 1xe2x80x94various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure).
As the HIV-1 epidemic continues to spread world wide, the need for an effective vaccine remains urgent. Efforts to develop such a vaccine have been hampered by three main factors: (a) the extraordinary ability of the virus to mutate; (b) inability of most known specificities of anti-HIV antibodies to neutralise HIV primary isolates consistently; and (c) lack of understanding of the correlates of protective immunity to HIV infection. In view of the complex biology of HIV-host interactions, the most fruitful avenue may be development of multivalent HIV immunogens tailored to HIV isolates in specific geographical locations.
CD8 CTL that kill HIV-infected cells and antibodies that broadly neutralise HIV primary isolates might be protective anti-HIV immune responses in uninfected individuals who are subsequently exposed to HIV (ref. 2).
The definition of a successful preventative HIV immunogen is controversial. Protective anti-HIV immune responses may prevent HIV infection completely, may allow only transient infection, leading to clearance of virus, or may merely limit the extent of HIV infection, but in so doing prevent the development of AIDS. One suggestion is that clearance of HIV occasionally occurs after both maternal-fetal HIV transmission (ref. 3) and sexual transmission of HIV (ref. 4). Consequently, if protective anti-HIV immune responses could be induced by an immunogen in an HIV-uninfected person, protection might be achieved via early termination of HIV infection.
It has been shown that anti-recombinant (r) gp120 envelope antibodies raised in animals or in human volunteers neutralise HIV grown in laboratory-adapted T-cell lines but not primary isolates of the virus grown in peripheral blood mononuclear cells. This observation raises important questions about the roles of various spcificities of neutralising antibodies in protection against HIV. The predominant types of anti-HIV neutralising antibodies raised against gp120 are antibodies against the third variable (V3) region of gp120, as well as antibodies against the conformationally determined CD4 binding site centred around the fourth constant (C4) region of gp120. Although laboratory-adapted variants are pathogenic and have caused AIDS in man after laboratory accidents (ref. 5), the relevance of these variants in vivo in community-acquired infections is unknown. Serum concentrations of antibodies against the V3 gp120 region and of antibodies that neutralise laboratory-adapted HIV strains do not protect individuals from developing AIDS (ref. 6), nor do anti-V3 antibodies seem protective against maternal-fetal HIV transmission (ref. 7).
Thus, for induction by HIV immunogens of neutralising antibodies to prevent HIV infection, HIV immunogens are probably needed which are capable of inducing anti-HIV antibodies that neutralise both HIV laboratory-adapted isolates and HIV primary isolates grown in peripheral blood mononuclear cells (ref. 8).
There is suggestive evidence that envelope oligomers of HIV primary isolates may be appropriate immunogens for induction of anti-HIV neutralising antibodies against primary HIV isolates grown in peripheral blood mononuclear cells. Future studies are expected to focus on the envelope of HIV primary isolates as the target of neutralising antibodies. If HIV envelope oligomers are successful in inducing antibodies that neutralise HIV primary isolates, the neutralising antibody specificity may be variant specific and, if so, the issue of HIV variability would still need to be addressed.
Several candidate vaccines, based on different concepts, are at different stages in the HIV vaccine development pipeline. Candidate vaccines based on the subunit recombinant envelope concept and produced in mammalian cells have been shown to protect chimpanzees from HIV-1 infection and to be safe and reasonably immunogenic in humans, inducing neutralizing antibodies. A second generation of candidate vaccines, which are based on live vectors expressing the envelope and other HIV-1 genes, and which are capable of inducing CTLs are beginning to be evaluated in human trials. Newer generations of candidate vaccines now being mostly explored in animal experiments are using combinations of subunit recombinant proteins or live vectored vaccines with other immunogens, such as synthetic peptides or pseudovirions, or are based on more novel approaches, including nucleic acid immunization and perhaps whole-inactivated or live attenuated vaccines.
However, there is a clear need for immunogenic preparations incorporating antigens or antigen fragments from primary or clinical HIV isolates. These preparations will be useful as vaccine candidates, as antigens in diagnostic assays and kits and for the generation of immunological reagents for diagnosis of HIV and other retroviral disease and infection.
Particular prior art immunogenic preparations include non-infectious, non-replicating HIV-like particles. PCT applications WO 93/20220 published Oct. 14, 1993 and WO 91/05860 published May 2, 1990 (Whitehead Institute for Biomedical Research), teach constructs comprising HIV genomes having an alteration in a nucleotide sequence which is critical for genomic RNA packaging, and the production of non-infectious immunogenic HIV particles produced by expression of these constructs in mammalian cells.
PCT application WO 91/07425 published May 30, 1991 (Oncogen Limited Partnership) teaches non-replicating retroviral particles produced by coexpression of mature retroviral core and envelope structural proteins such that the expressed retroviral proteins assemble into budding retroviral particles. A particular non-replicating HIV-1 like particle was made by coinfecting mammalian host cells with a recombinant vaccinia virus carrying the HIV-1 gag and protease genes and a recombinant vaccinia virus carrying the HIV-1 env gene.
In published PCT application WO 91/05864 in the name of the assignee hereof (which is incorporated herein by reference thereto), and corresponding granted U.S. Pat. Nos. 5,439,809 and 5,571,712, there is described particular non-infectious non-replicating retrovirus-like particles containing at least gag, pol and env proteins in their natural conformation and encoded by a modified retroviral genome deficient in long terminal repeats and containing gag, pol and env genes in their natural genomic arrangement.
In WO 96/06177 and corresponding copending U.S. patent application Ser. No. 08/292,967 filed Aug. 22, 1994 (abandoned), assigned to the assignee hereof and the disclosures of which are incorporated herein by reference, there are described further mutations to the HIV genome of the constructs of U.S. Pat. Nos. 5,439,809 and 5,571,712 to reduce gag-dependent RNA packaging of the HIV-1 genome, to eliminate reverse transcriptase activity of the pol gene product, to eliminate integrase activity of the pol gene product and to eliminate RNAse activity of the pol gene product, through genetic manipulation of the gag and pol genes.
In the preferred vectors described in the aforementioned U.S. Pat. Nos. 5,439,809 and 5,571,712 and U.S. application Ser. No. 08/292,967, abandoned, a metallothionein promoter is employed, which requires the addition of an inducer for expression to be effected. The use of such promoters for commercial scale production of such HIV-like particles is impractical, in view of the cost of the heavy metals employed and the toxic effect of such heavy metals on the expression cells.
It is desirable, therefore, to employ a constitutive promoter for expression of the HIV-like particles. However, it has been found that substitution of a constitutive promoter, results in cell toxicity, limiting the useful period of induction of the HIV-like particles.
It has now been surprisingly found that, by effecting specific genetic modification to the HIV genome, as set forth herein, it is possible to effect long term constitutive expression of non-infectious, non-replicating, immunogenic HIV-like particles without causing any toxic effect on the mammalian cells expressing the particles.
In accordance with one aspect of the present invention, there is provided a nucleic acid molecule, comprising a modified HIV genome devoid of long terminal repeats and wherein vpr and tat sequences are functionally disabled and a constitutive promoter operatively connected to the modified HIV genome for constitutive expression of the modified genome to produce non-infectious, non-replicating and immunogenic HIV-like particles.
The vpr and tat sequences may be functionally disabled by the insertion of stop codons therein preventing expression of the respective encoded gene products.
The HIV genome may be further modified by replacing the signal peptide of gp120 by a gp120 expression enhancing sequence, specifically the signal peptide encoding sequence of glycoprotein D (gD) of herpes simplex virus (HSV).
In a preferred embodiment of the invention, the env gene in the nucleic acid molecule encodes a env gene product from a primary HIV-1 isolate.
In accordance with the aforementioned U.S. application Ser. No. 08/292,967, abandoned, (WO 96/06177), the HIV genome of the nucleic acid molecule may be further modified to effect reduction in gag-dependent RNA packaging of the gag gene product. Such reduction in gag-dependent RNA packaging of the gag gene product may be effected by replacing Cys 395 and Cys 395 of the gag gene product of HIV-1 LAI isolate, or the corresponding amino acids of another isolate, by serine.
In addition or alternatively, also in accordance with U.S. application Ser. No. 08/292,967, abandoned, (WO 96/06177), the HIV genome of the nucleic acid molecule may be further modified to substantially eliminate reverse transcriptase activity, integrase activity and RNAse activity. In this regard, a BalI-BalI portion of the pol gene may be deleted between nucleotides 2655 and 4507 of the LAI isolate of HIV-1 or the corresponding portion of the pol gene of another HIV-1 isolate.
The constitutive promoter employed herein may be the human immediate early cytomegalovirus promoter or any other convenient constitutive promoter of expression of the non-infectious, non-replicating immunogenic HIV-like particles in mammalian cells. An expression enhancing sequence may be provided between the promoter and the modified genomes. Such an expression enhancing sequence may be the human cytomegalovirus Intron A sequence.
For the purposes of expression of the HIV-like particles, the nucleic acid molecule provided herein may be incorporated into an expression vector, which may be one having the identifying characteristics of plasmid pCMVgDtatxe2x88x92vprxe2x88x92, as described in detail below.
The present invention, in another aspect thereof, provides a method of obtaining a non-infectious, non-replicating, immunogenic HIV-like particle, which comprises incorporating into an expression vector a nucleic acid molecule comprising a modified HIV genome devoid of long terminal repeats and wherein vpr and tat sequences are functionally disabled and a constitutive promoter operatively connected to the modified HIV genome, introducing the expression vector into mammalian cells, and constitutively expressing the nucleic acid molecule in the cells to stably produce non-infectious, non-replicating, immunogenic HIV-like particles.
The nucleic acid molecule incorporated into the expression vector may have the various features discussed above with respect to the nucleic acid molecule aspect of the invention. In addition to constitutive expression, expression of the nucleic acid molecule may also be enhanced by employing chemical agents which enhance the specific promoter employed. The expression vector preferably is one having the identifying characteristics of plasmid pCMVgDtatxe2x88x92vprxe2x88x92.
Such method enables the HIV-like particles to be produced over a long term without any adverse toxic effect on the mammalian cells. As seen below, applicants have maintained constitutive expression through more than 50 passages of the cells. The present invention extends to the non-infectious, non-replicating immunogenic HIV-like particles lacking Tat and Vpr and producible by the method aspect of the invention as well as immunogenic compositions comprising the same and methods of immunization using such compositions.
The non-infectious, non-replicating immunogenic HIV-like particles produced according to the procedure of the invention may be employed in immunogenic compositions, as described in the aforementioned U.S. Pat. Nos. 5,439,809 and 5,571,712 and U.S. patent application Ser. No. 08/292,967, abandoned (WO 96/06177), for inducing an immune response in a host.
Advantages of the present invention include the ability to effect long-term production of non-infectious, non-replicating immunogenic HIV-like particles, thereby providing an expression system for such particles which is more useful in commercial production of the HIV-like particles than the systems previously considered.